Ciliary neurotrophic factor (CNTF) is a protein that is required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliary ganglion is anatomically located within the orbital cavity, lying between the lateral rectus and the sheath of the optic nerve; it receives parasympathetic nerve fibers from the oculomotor nerve which innervates the ciliary muscle and sphincter pupillae.
Over the past decade, a number of biological effects have been ascribed to CNTF in addition to its ability to support the survival of ciliary ganglion neurons. CNTF is believed to induce the differentiation of bipotential glial progenitor cells in the perinatal rat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73). Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39-46). In addition, CNTF supports the survival and differentiation of motor neurons, hippocampal neurons and presympathetic spinal cord neurons [Sendtner, et al., 1990, Nature 345: 440-441; Ip, et al. 1991, J. Neurosci. 11:3124-3134; Blottner, et al. 1989, Neurosci. Lett. 105:316-320].
It has long been known that innervation of skeletal muscle plays a critical role in the maintenance of muscle structure and function. Skeletal muscle has been shown recently to be a target of positive CNTF actions. Specifically, CNTF prevents both the denervation-induced atrophy (decreased wet weight and myofiber cross sectional area) of skeletal muscle and the reduced twitch and tetanic tensions of denervated skeletal muscle (Helgren et al., Cell 76:493-504 (1994)). In this model, human CNTF also produces an adverse effect that is manifested as a retardation of weight gain. This adverse effect has also been observed in clinical trials with rHCNTF for the treatment of ALS. Therefore, simultaneous measurements of muscle weight and animal body weight following denervation could be used as a measure of efficacy and adverse reaction, respectively, in response to treatment with rHCNTF or other compounds. The ratio of the potency values obtained from these measurements is defined as the therapeutic index (T.I.), expressed here as TD25/ED50, so that the higher the value of T.I., the safer the compound at a therapeutic dose.
CNTF has been cloned and synthesized in bacterial expression systems, as described by Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in International Publication No. WO 91/04316, published on Apr. 4, 1991, which are incorporated by reference in their entirety herein.
The receptor for CNTF (termed xe2x80x9cCNTFRxcex1xe2x80x9d) has been cloned, sequenced and expressed [see Davis, et al. (1991) Science 253:59-63]. CNTF and the haemopoetic factor known as leukemia inhibitory factor (LIF) act on neuronal cells via a shared signaling pathway that involves the IL-6 signal transducing component gp130 as well as a second, ,xcex2-component (know as LIFR xcex2); accordingly, the CNTF/CNTF receptor complex can initiate signal transduction in LIF responsive cells, or other cells which carry the gp130 and LIFRxcex2 components [Ip, et al. (1992) Cell 69:1121-1132].
In addition to human CNTF, the corresponding rat (Stxc3x6ckli et al., 1989, Nature 342:920-923), and rabbit (Lin et al., 1989, J. Biol. Chem. 265:8942-8947) genes have been cloned and found to encode a protein of 200 amino acids, which share about 80% sequence identity with the human gene. Both the human and rat recombinant proteins have been expressed at exceptionally high levels (up to 70% of total protein) and purified to near homogeneity.
Despite their structural and functional similarity, recombinant human and rat CNTF differ in several respects. The biological activity of recombinant rat CNTF in supporting survival and neurite outgrowth from embryonic chick ciliary neurons in culture is four times better than that of recombinant human CNTF [Masiakowski et al., (1991), J. Neurochem. 57:1003-1012]. Further, rat CNTF has a higher affinity for the human CNTF receptor than does human CNTF.
A surprising difference in the physical properties of human and rat CNTF, which are identical in size, is their different mobility on SDS gels. This difference in behaviour suggests the presence of an unusual structural feature in one of the two molecules that persists even in the denatured state (Masiakowski et al., 1991, id.).
Mutagenesis by genetic engineering has been used extensively in order to elucidate the structural organization of functional domains of recombinant proteins. Several different approaches have been described in the literature for carrying out deletion or substitution mutagenesis. The most successful appear to be alanine scanning mutagenesis [Cunningham and Wells (1989), Science 244: 1081-1085] and homolog-scanning mutagenesis [Cunningham et al., (1989), Science 243:1330-1336]. These approaches helped identify the receptor binding domains of growth hormone and create hybrid proteins with altered binding properties to their cognate receptors.
To better understand the physical, biochemical and pharmacological properties of rHCNTF, applicant undertook rational mutagenesis of the human and rat GNTF genes based on the different biological and physical properties of their corresponding recombinant proteins (See Masiakowski, P., et al. (1991) J. Neurochem., 57, 1003-1012). Applicant has found that the nature of the amino acid at position 63 could greatly enhance the affinity of human CNTF for sCNTFRxcex1 and its biological potency in vitro (Panayotatos, N., et al., J. Biol. Chem., 268, 19,000-19,003 (1993); Panayotatos, N., et al., Biochemistry, 33, 5813-5818 (1994).
An object of the present invention is to provide novel CTNF-related neurotrophic factors for the treatment of diseases or disorders including, but not limited to, motor neuron diseases and muscle degenerative diseases.
A further object of the present invention is to provide a method for identifying CNTF-related factors, other than those specifically described herein, that have improved therapeutic properties.
These and other objects are achieved in accordance with the invention, whereby amino acid substitutions in human CNTF protein enhance its therapeutic properties. In one embodiment, alterations in electrophoretic mobility are used to initially screen potentially useful modified CNTF proteins.
In a preferred embodiment, the amino acid glutamine in position 63 of human ONTF is replaced with arginine (referred to as 63QR) or another amino acid which results in a modified CNTF molecule with improved biological activity. In further embodiments, rHONTF variants combine the 63QR mutation with three other novel features:
1) Deletion of the last 13 amino acid residues (referred to as xcex94C13) to confer greater solubility to rHCNTF without impairing its activity;
2) Substitution of the unique cysteine residue at position 17, which results in stabilization of rHCNTF in physiological buffer, at physiological pH and temperature conditions without affecting its activity; or
3) Substitution of amino acid residue 64W, which alters the biological activity of rHCNTF in vitro and which results in a 7-fold improvement of its therapeutic index in vivo.
In another preferred embodiment, a molecule designated RG297 (rHCNTF,17CA63QRxcex94C13) combines a 63QR substitution (which confers greater biological potency) with a deletion of the terminal 13 amino acid residues (which confers greater solubility under physiological conditions) and a 17CA substitution (which confers stability, particularly under physiological conditions at 37xc2x0 C.) and shows a 2-3 fold better therapeutic index than rHCNTF in an animal model.
In another preferred embodiment, a molecule designated RG242 is described that carries the double substitution 63QR64WA which results in a different spectrum of biological potency and a 7-fold higher therapeutic index.
In another preferred embodiment, a molecule designated RG290 is described that carries the double substitution 63QRxcex94C13 which confers greater solubility under physiological conditions.